Method for synchronously and instantly detecting multiple respiratory pathogens

Abstract

The invention provides a method for synchronously and instantly detecting various respiratory pathogens, a bipolar electrode array chip adopted in detection comprises a plurality of detection areas and a plurality of amplification areas, an isothermal amplification technology and electrochemical luminescence detection are integrated, RGB values of pictures are subjected to data analysis, and if an ECL signal quenching phenomenon occurs in a certain detection area, the detection area is judged to be a certain detection area, and if the ECL signal quenching phenomenon occurs in the certain detection area, the detection area is judged to be a certain detection area. If the ECL signal appearing in a certain detection area and the ECL signal appearing in the detection area corresponding to the negative control sample are not obviously changed or slightly enhanced, the to-be-detected sample corresponding to the detection area is judged to be a negative sample, and if the ECL signal appearing in the detection area and the ECL signal appearing in the detection area corresponding to the negative control sample are not obviously changed or slightly enhanced, the to-be-detected sample corresponding to the detection area is judged to be a positive sample. And multi-target synchronous on-site rapid high-sensitivity detection of respiratory pathogens can be realized.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, in particular to the field of molecular diagnosis, and specifically to a method for simultaneous and instant detection of multiple respiratory pathogens. Background Art

[0002] Real-time fluorescence quantitative PCR (qPCR) is the gold standard for clinical detection of respiratory pathogens, but it requires professional personnel, complex operations, and large instruments, which is not suitable for on-site detection. In order to overcome the limitations of the above methods, a portable electrochemiluminescence detection device has been developed. The combination of an intelligent portable device with a CCD camera and an electrochemiluminescence detection component is conducive to on-site detection. Specifically, an intelligent portable device with a CCD camera is installed on the optical dark box of the electrochemiluminescence detection component. The intelligent portable device can be a smart phone. An electrochemiluminescence reaction occurs in the optical dark box, and the ECL signal is collected by shooting with a CCD camera. The intelligent portable device then analyzes the collected signal to obtain the test result.

[0003] Based on the existing technology, a more sensitive detection method is needed to achieve rapid and accurate on-site simultaneous multi-target detection of respiratory pathogens. Summary of the invention

[0004] The present invention provides a method for simultaneous and instant detection of multiple respiratory pathogens, integrating isothermal amplification technology (LAMP) and electrochemiluminescence detection, and can achieve multi-target simultaneous on-site rapid and highly sensitive detection of respiratory pathogens.

[0005] Before describing the scheme of the present invention, the following English abbreviations are explained: ECL: electrochemiluminescence detection signal, LAMP: loop-mediated isothermal amplification technology, ABS: acrylonitrile butadiene styrene plastic sheet, ITO: indium tin oxide, [Ru(bpy) 3 ] 2+ : terpyridine ruthenium, TPrA: tri-n-propylamine, PDMS: polydimethylsiloxane, MB: methylene blue.

[0006] The technical solution provided by the present invention is:

[0007] A method for simultaneous instant detection of multiple respiratory pathogens, comprising:

[0008] S1. Making a bipolar electrode array chip, the bipolar electrode array chip comprising a plurality of detection areas and a plurality of amplification areas;

[0009] S2. Setting a stable and continuous temperature to ensure isothermal amplification LAMP in the amplification area of ​​the bipolar electrode array chip;

[0010] S3. The detection area of ​​the bipolar electrode array chip generates an electrochemiluminescent signal, and the smart portable device with a CCD camera takes a picture of the electrochemiluminescent signal;

[0011] S4. Perform data analysis on the RGB values ​​of the image. If ECL signal quenching occurs in a certain detection area, the sample to be tested corresponding to the detection area is judged to be a positive sample. If the ECL signal appearing in a certain detection area has no obvious change or is slightly enhanced compared with the ECL signal appearing in the detection area corresponding to the negative control sample, the sample to be tested corresponding to the detection area is judged to be a negative sample.

[0012] The preparation of the bipolar electrode array chip comprises the following steps:

[0013] (1) etching the middle of the first ITO electrode to disconnect the electrodes on both sides of the first ITO electrode;

[0014] (2) attaching an ABS plastic sheet to the first ITO electrode, wherein a plurality of detection areas and a plurality of amplification areas are provided on the ABS plastic sheet, wherein the detection area and the amplification area are respectively located on one side of the first ITO electrode, adding a negative control sample to one amplification area, and adding the test samples to the other amplification areas, and adding the LAMP amplification system and the electrochemical redox probe MB to the amplification areas to which the test samples are added, and adding the electrochemical luminescence reaction system to the detection area;

[0015] (3) covering a plurality of second ITO electrodes on the ABS plastic sheet, and each second ITO electrode connects an amplification zone and a detection zone in series to obtain a bipolar electrode array chip;

[0016] Moreover, the bipolar electrode array chip is installed in the optical dark box of the electrochemical luminescence detection component. The specific installation process is: a pair of chip racks are provided in the optical dark box, each chip rack is equipped with a conductive sheet, the conductive sheet is electrically connected to the power supply module of the electrochemical luminescence detection component, one chip rack fixes one side of the bipolar electrode array chip, and the conductive layer and the conductive sheet of the bipolar electrode array chip are in contact and electrically connected to each other.

[0017] In the present invention, when the sample to be tested is positive, the amplification area of ​​the bipolar electrode array chip contains a LAMP amplification system and an electrochemical redox probe MB. Since MB can be strongly embedded in a large number of double-stranded structures generated by LAMP amplification, this result will cause the apparent electron transfer capacity of the electrode to decrease, thereby causing a decrease in the Faraday current, and ultimately causing the electrochemical luminescence signal in the detection area of ​​the bipolar electrode array chip to be quenched, thereby achieving high-sensitivity detection.

[0018] Furthermore, the present invention uses a bipolar electrode array chip to detect multiple samples, thereby achieving simple and rapid simultaneous detection of multiple respiratory targets.

[0019] In addition, because the conductive sheet of the chip holder directly contacts the conductive layer of the bipolar electrode array chip, it effectively isolates the interference of the external environment on the electrode, avoids the influence of potential fluctuation on the electrochemical luminescence detection results, and ensures the uniformity and stability of the potential output. Therefore, the design of the chip holder can ensure that a uniform potential is provided for the bipolar electrode array chip, thereby improving the detection accuracy.

[0020] Furthermore, ruthenium terpyridine ([Ru(bpy) 3 ] 2+ ) and tri-n-propylamine (TPrA) electrochemiluminescence reaction system.

[0021] Preferably, MB and [Ru(bpy) 3 ] 2+ When the concentrations were 0.1 μM and 0.4 mM, respectively, better detection strategy effects could be achieved.

[0022] Furthermore, the chip frame includes a pair of clips, the conductive sheet is arranged in the pair of clips, and one clip clamps one side of the bipolar electrode array chip.

[0023] Preferably, the clamping tightness of the clamp is adjusted by a bolt structure.

[0024] Furthermore, setting the temperature to 65° C. and maintaining it for 40 minutes is a preferred condition to ensure isothermal amplification LAMP in the amplification region of the bipolar electrode array chip. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the preparation process of the bipolar electrode array chip in Example 1 of the present invention;

[0026] Figure 2 is a structural appearance diagram of a portable electrochemiluminescence detection device in Example 2 of the present invention;

[0027] Figure 3 yes Figure 2 The schematic diagram of the assembly of the portable electrochemiluminescence detection device shown;

[0028] Figure 4 This is the experimental result of detecting clinical samples in Example 2 of the present invention. DETAILED DESCRIPTION

[0029] Example 1 Preparation of bipolar electrode array chip

[0030] Reference Figure 1 , the preparation process of the bipolar electrode array chip includes:

[0031] Step 1: Etch the middle of a first ITO electrode 1 with a size of 4×4×0.11 cm and a square resistance of 70-90Ω / □ to disconnect the left and right electrodes of the first ITO electrode 1, that is, the current on both sides cannot be conducted.

[0032] Step 2: Place a 3.2×4cm, 1mm thick, double-sided adhesive, and 10 holes ABS plastic plate 2 tightly against the etched first ITO electrode. The 5 holes on the left half of the ABS plastic plate 2 are the detection area, and the 5 holes on the right half are the amplification area. The detection area and the amplification area are located on the left and right sides of the first ITO electrode 1, respectively.

[0033] Step 3: Add a negative control sample to one amplification zone, add the test sample to the other amplification zones, and add 25 μl of LAMP amplification system and 0.1 μM electrochemical redox probe MB to the amplification zones with the test sample, and add 0.4 mM [Ru(bpy) 3 ] 2+ and 1000mM TPrA electrochemiluminescence reaction system.

[0034] Step 4: Cover the ABS plastic sheet 2 with 5 second ITO electrodes 3 with a size of 0.5x3x0.11cm and a square resistance of 70-90Ω / □. Each second ITO electrode 3 connects an amplification area and a detection area in series to obtain a bipolar electrode array chip 4.

[0035] The surface of the bipolar electrode array chip 4 is coated with an anti-pollution layer of polydimethylsiloxane to prevent external pollutants from interfering with the detection results and also to prevent contamination between samples.

[0036] The LAMP amplification system is shown in Table 1 below, wherein FIP and BIP are inner primers corresponding to respiratory pathogens, F3 and B3 are outer primers, Loop F and Loop B are loop primers, dNTPs are deoxyribonucleoside triphosphates, and target is nucleic acid extracted from nasopharyngeal swabs corresponding to respiratory pathogens.

[0037] Table 1 LAMP amplification system

[0038]

[0039] Example 2: Simultaneous and immediate detection of multiple respiratory pathogens

[0040] like Figure 2 and Figure 3As shown, the portable electrochemiluminescence detection device used in the embodiment of the present invention includes: a smart phone 5 with a CCD camera, an adapter 6 fixing the smart phone 5 on an optical dark box 7, a circuit board 8, a temperature control board 9 and a lithium battery 10 are all fixed in the optical dark box 7, and a pair of chip clips 12 are also provided on the base 11 of the optical dark box 7.

[0041] The lithium battery 10 is connected to the circuit board 8 and the temperature control board 9, and the circuit board 8 is connected to the chip clip 12. In the embodiments of the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral one; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the internal connection of the two elements or the interaction relationship between the two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to the specific circumstances. The circuit board 8 can provide the voltage required for the electrochemical luminescence signal, the temperature control board 9 can provide the temperature required for the isothermal amplification of LAMP, and the lithium battery 10 can provide the working voltage of the circuit board 8 and the temperature control board 9. A chip frame 12 includes a pair of clips 13, a copper conductive sheet 14 is arranged in the pair of clips 13, the pair of clips 13 clamps one side of the bipolar electrode array chip 4, and the pair of clips 13 is used to adjust the degree of clamping by a bolt structure 15, the bipolar electrode array chip 4 and the copper conductive sheet 14 are tightly attached together and electrically connected to each other. The bipolar electrode array chip 4 is prepared by Example 1.

[0042] Figure 1 Step 4 also shows the connection between the chip frame 12 and the bipolar electrode array chip 4. Of course, the bipolar electrode array chip 4 is installed on the chip frame 12 only when it needs to be tested.

[0043] based on Figure 2 and Figure 3 The portable electrochemiluminescence detection device can be used to detect multiple respiratory pathogens simultaneously, including:

[0044] The temperature control plate 9 is started, and the temperature control plate 9 controls the heating plate 16 to slowly heat to the specified temperature of 65 degrees Celsius. The heating plate 16 is connected to the bipolar electrode array chip 4. After 40 minutes of heating, the temperature control plate 9 is turned off, and isothermal amplification LAMP occurs in the amplification area of ​​the bipolar electrode array chip 4.

[0045] The parameters of the CCD camera used by the smartphone 5 are set, such as exposure, color, contrast, exposure time, focal length, etc. The circuit board 8 outputs the set output voltage of 4.8V, and the detection area of ​​the bipolar electrode array chip 4 undergoes an electrochemical luminescence reaction to generate an electrochemical luminescence signal. The smartphone 5 takes a photo and uploads the photo to the cloud. The cloud automatically processes the photo and sends the extracted RGB value to the smartphone 5.

[0046] Smart phone 5 performs data analysis on the RGB values ​​of the image. If ECL signal quenching occurs in a certain detection area, the sample to be tested corresponding to the detection area is judged to be a positive sample. If the ECL signal appearing in a certain detection area has no obvious change or is slightly enhanced from the ECL signal appearing in the detection area corresponding to the negative control sample, the sample to be tested corresponding to the detection area is judged to be a negative sample.

[0047] Figure 4 This is the experimental result of using the detection method of Example 2 for 4 clinical samples suspected of suffering from influenza A virus. The detection area of ​​the bipolar electrode array chip 4 undergoes an electrochemical luminescence reaction to generate a red electrochemical luminescence signal. After the smart phone 5 uploads the photographed photo to the cloud, the cloud extracts the RGB value of the red electrochemical luminescence signal. Among them, No. 1 is a negative control sample, and Nos. 2, 3, 4, and 5 are clinical samples suspected of suffering from influenza A virus, and their RGB values ​​are 68.75, 63.41, 33.91, 40.35, and 38.49, respectively. Therefore, the ECL signal of sample No. 2 has no obvious change, and the ECL signals of samples Nos. 3, 4, and 5 are quenched. Therefore, it is judged that sample No. 2 is a negative sample for influenza A, and samples Nos. 3, 4, and 5 are positive samples for influenza A.

[0048] Example 2 achieves simultaneous and instant detection of multiple respiratory pathogens. The electrochemiluminescence detection technology combined with the LAMP amplification technology and the chip clip design all improve the sensitivity and accuracy of the detection.

[0049] The embodiments of the present invention are described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Various changes can be made within the knowledge scope of ordinary technicians in the technical field without departing from the purpose of the present invention.

Claims

1. A method for simultaneous and immediate detection of multiple respiratory pathogens, characterized in that: include: S1. Making a bipolar electrode array chip, the bipolar electrode array chip comprising a plurality of detection areas and a plurality of amplification areas; S2. Setting a stable and continuous temperature to ensure isothermal amplification LAMP in the amplification area of ​​the bipolar electrode array chip; S3. The detection area of ​​the bipolar electrode array chip generates an electrochemiluminescent signal, and the smart portable device with a CCD camera takes a picture of the electrochemiluminescent signal; S4. Perform data analysis on the RGB values ​​of the image. If ECL signal quenching occurs in a certain detection area, the sample to be tested corresponding to the detection area is judged to be a positive sample. If the ECL signal appearing in a certain detection area has no obvious change or is slightly enhanced from the ECL signal appearing in the detection area corresponding to the negative control sample, the sample to be tested corresponding to the detection area is judged to be a negative sample. The preparation of the bipolar electrode array chip comprises the following steps: (1) etching the middle of the first ITO electrode to disconnect the electrodes on both sides of the first ITO electrode; (2) attaching an ABS plastic sheet to the first ITO electrode, wherein a plurality of detection areas and a plurality of amplification areas are provided on the ABS plastic sheet, wherein the detection area and the amplification area are respectively located on one side of the first ITO electrode, adding a negative control sample to one amplification area, and adding the test samples to the other amplification areas, and adding the LAMP amplification system and the electrochemical redox probe MB to the amplification areas to which the test samples are added, and adding the electrochemical luminescence reaction system to the detection area; (3) covering a plurality of second ITO electrodes on the ABS plastic sheet, and each second ITO electrode connects an amplification zone and a detection zone in series to obtain a bipolar electrode array chip; Moreover, the bipolar electrode array chip is installed in the optical dark box of the electrochemical luminescence detection component. The specific installation process is: a pair of chip racks are provided in the optical dark box, each chip rack is equipped with a conductive sheet, the conductive sheet is electrically connected to the power supply module of the electrochemical luminescence detection component, one chip rack fixes one side of the bipolar electrode array chip, and the conductive layer and the conductive sheet of the bipolar electrode array chip are in contact and electrically connected to each other.

2. The method for simultaneous and immediate detection of multiple respiratory pathogens according to claim 1, characterized in that: The component added to the detection zone is [Ru(bpy)3] 2+ and TPrA electrochemiluminescence reaction system.

3. The method for simultaneous and immediate detection of multiple respiratory pathogens according to claim 2, characterized in that: MB and [Ru(bpy)3] 2+ The concentrations were 0.1 μM and 0.4 mM, respectively.

4. The method for simultaneous and immediate detection of multiple respiratory pathogens according to claim 1, characterized in that: The chip frame comprises a pair of clips, in which the conductive sheet is arranged, and one clip clamps one side of the bipolar electrode array chip.

5. The method for simultaneous and immediate detection of multiple respiratory pathogens according to claim 4, characterized in that: The clamping tightness of the clamp is adjusted by a bolt structure.

6. The method for simultaneous and immediate detection of multiple respiratory pathogens according to claim 1, characterized in that: Step S2, setting the temperature to 65°C and maintaining it for 40 minutes.

Images